title: Modulation of M(2) muscarinic receptor-receptor interaction by immunoglobulin G antibodies from Chagas' disease patients.
abstract: Circulating immunoglobulin (Ig)G antibodies against M(2) muscarinic acetylcholine receptors (M(2) mAChR) have been implicated in Chagas' disease (ChD) pathophysiology. These antibodies bind to and activate their target receptor, displaying agonist-like activity through an unclear mechanism. This study tested the ability of serum anti-M(2) mAChR antibodies from chronic ChD patients to modulate M(2) muscarinic receptor-receptor interaction by bioluminescence resonance energy transfer (BRET). Human embryonic kidney (HEK) 293 cells co-expressing fusion proteins M(2) mAChR-Renilla luciferase (RLuc) and M(2) mAChR-yellow fluorescent protein (YFP) were exposed to the serum IgG fraction from ChD patients, and BRET between RLuc and YFP was assessed by luminometry. Unlike serum IgG from healthy subjects and conventional muscarinic ligands, ChD IgG promoted a time- and concentration-dependent increase in the BRET signal. This effect neither required cellular integrity nor occurred as a consequence of receptor activation. Enhancement of M(2) receptor-receptor interaction by ChD IgG was receptor subtype-specific and mediated by the recognition of the second extracellular loop of the M(2) mAChR. The monovalent Fab fragment derived from ChD IgG was unable to reproduce the effect of the native immunoglobulin. However, addition of ChD Fab in the presence of anti-human Fab IgG restored BRET-enhancing activity. These data suggest that the modulatory effect of ChD IgG on M(2) receptor-receptor interaction results from receptor cross-linking by bivalent antibodies.
authors.affiliation: Laboratorio de Farmacología Molecular, Centro de Estudios Farmacológicos y Botánicos (CEFYBO-CONICET-UBA).
authors.forename: S P
authors.initials: SP
authors.lastname: Beltrame
authors.forename: S R
authors.initials: SR
authors.lastname: Auger
authors.forename: C R
authors.initials: CR
authors.lastname: Bilder
authors.forename: C I
authors.initials: CI
authors.lastname: Waldner
authors.forename: J C
authors.initials: JC
authors.lastname: Goin
mesh_terms: D000595:Amino Acid Sequence; D000913:Antibodies, Protozoan / Q000276:immunology*; D000918:Antibody Specificity; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics; D014355:Chagas Disease / Q000276:immunology* / Q000503:physiopathology; D018678:Cholinergic Agents / Q000494:pharmacology; D004735:Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology*; D049410:Luciferases, Renilla / Q000032:analysis / Q000235:genetics; D049449:Luminescence; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D017434:Protein Structure, Tertiary; D020239:Receptor Cross-Talk; D043585:Receptor, Muscarinic M2 / Q000187:drug effects / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000276:immunology; D014349:Trypanosoma cruzi / Q000276:immunology*
chemical_list: D000913:Antibodies, Protozoan; D001426:Bacterial Proteins; D018678:Cholinergic Agents; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D008164:Luminescent Proteins; D010446:Peptide Fragments; D043585:Receptor, Muscarinic M2; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049410:Luciferases, Renilla